PROJECT SUMMARY Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are large, multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. Mutations in the genes encoding mSWI/SNF complex subunits (i.e. ARID1A, SMARCA4, PBRM1, and SMARCB1) most often result in loss-of-function deletions of specific protein subunits, thus implicating their functions as critical tumor suppressors. In addition, other subunits such as SS18 undergo translocation, leading to cancer-specific fusion oncoproteins, such as the SS18-SSX fusion which binds to and hijacks mSWI/SNF complexes to new genomic sites. Finally, de novo, heterozygous mutations of ARID1B, SMARCB1 and ACTL6A are often linked to intellectual disability and autism spectrum disorders. Therefore, there is an urgent need to understand the mechanisms underlying mSWI/SNF complex perturbation in order to devise new, on-target therapeutic strategies for the now over 20% of human cancers and other diseases linked to mSWI/SNF disruption. However, the structure and the mechanism of action of these crucial chromatin regulators remains largely unknown. To begin to address this, we have developed a series of orthogonal approaches to define the assembly pathway and modular organization (functional and structural modules) of SWI/SNF complexes both in vitro and in vivo. We recently established a highly robust method to purify all three types of mammalian SWI/SNF complexes: canonical BAF, PBAF, and a newly defined complex termed ncBAF. Using CRISPR-Cas9-based knockout (KO) approaches we have worked to generate a library of subunit KO mutant cell lines, spanning the three groups of SWI/SNF complexes, to define subunit requirements for assembly and function. Having established these foundational studies and scientific premise, herein we propose to: (1) define the mechanism of assembly and modular architecture of endogenously-purified mSWI/SNF complexes; (2) determine mSWI/SNF targeting and function on chromatin using a combination of complex purification with DNA- barcoded nucleosome libraries and ChIP-seq using antibodies designed to complex-specific subunit epitopes; (3) determine the 3D structure of endogenous mammalian SWI/SNF complexes using cryo-EM and x-ray crystallography. Successful completion of these studies will lead to the establishment of the first comprehensive structural map of the mSWI/SNF complex family and will provide new insights regarding complex mechanism of action across diverse chromatin landscapes in normal and disease settings. Importantly, the multidisciplinary investigations proposed, active mentorship, and career development activities will provide a uniquely strong foundation for my transition to independence and productive young career as an assistant professor.